FIG. 3.

Methylation status of p21^Cip1 promoter in human lung cancer cells. (A) Schematic diagram of the p21^Cip1 promoter. The CpG island of p21^Cip1 promoter (positions −313 to +552 relative to the transcription start site; National Center for Biotechnology Information [NCBI] no. U24170) was determined by computer program (WebGene [http://www.itba.mi.cnr.it/webgene/]). Primers were designed to amplify a fragment spanning positions −233 to +2 (235 bp, black box). Within this fragment there is one TaqI recognition site (TCGA, presented as T in the figure) and two HhaI recognition sites (GCGC, presented as H in the figure). (B and C) DNA from 16 human lung cancer cell lines and normal human blood cells was treated with bisulfite and then PCR amplified. The PCR products were purified and digested with the CG-containing enzymes HhaI (B) or TaqI (C). Digested samples were size separated by 8% PAGE. DNA not treated with bisulfite served as a negative control. DNA treated with Sss methylase was the positive control. Digested fragments correspond to methylated DNA.